The concentration of free cytokines or chemokines present in a normal biological fluid such as human plasma is near or below the detection limit of conventional ELISA assays. For example, it has been reported that a conventional ELISA assay whose detection limit is about 6 picomols (pM) cannot detect IL-8 from within normal human plasma (Leonard et al. (Document 1)). Enhancement of measurement sensitivity and reduction of the non-specific background associated with the biological fluid sample are chief problems to be solved in order to attain accurate measurement of chemokine concentration in a biological fluid sample.
In recent years, a time-resolved fluoroimmunoassay method which utilizes a europium complex has been developed, and is being used in clinical applications (Kropf et al., (Document 2)). The radiation wavelength (615 nm) of a free, complexed europium ion (Eu3+) is not influenced by the excitation wavelength (340 nm) or by a transient background fluorescence (350 to 600 nm) associated with a certain type of protein, which is convenient. One type of analysis method which is based on this principle is commercialized as DELFIA (dissociation-enhanced lanthanoid fluoroimmunoassay; Pharmacia), and is utilized in assays of TNF α and IL-6. However, DELFIA has not been successful in accurately measuring the concentration of such cytokines in plasma (Ogata et al. (Document 3)).
Recently, a group led by Matsumoto has developed a 4,4′-bis(1″,1″,2″,2″,3″,3″,-heptafluoro-4″,6″,-hexanedion-6″-yl)-sulpho-o-terphenyl(BHHCT)-Eu3+ complex as a labeling compound. This complex is capable of directly binding to proteins, and allows for highly sensitive analysis via a time-resolved type fluorescence measurement (Yuan et al. ('97)(Document 4) and Yuan et al. ('98)(Document 5)). BHHCT has a β-diketone structure, and has a binding stability constant with respect to Eu3+ as high as 1010M−1. A resultant Eu3+ complex exhibits quite excellent properties, as evidenced by a lifetime which exceeds 400 microseconds (μs), and absorption and emission wavelength maximals of 330 nm and 615 nm. This complex has been indicated to be useful for the detection of α-fetoprotein (Yuan et al. ('98)(Document 5) and immunoglobulin E (IgE) Yuan et al.('97)(Document 4)), which are tumor markers in human plasma. However, no instances are known in which such an Eu3+ complex has been applied to the detection of cytokines in a biological fluid sample.
Stromal cell-derived factor-1 (SDF-1) is a cytokine belonging to the chemokine family, which was first cloned from a stromal cell line in 1993 (Tashiro et al. (Document 6)). SDF-1 is a chief ligand for a CXCR4 receptor (Bleul et al. (Document 7) and Oberlin et al. (Document 8)). This receptor is known to function as a CD4 co-receptor for a subgroup of human immunodeficiency virus type 1 (HIV-1). Furthermore, recent study has shown that polymorphism of the SDF-1 gene is involved in slowing of the progression of acquired immunodeficiency syndrome (AIDS) (e.g., Winkler et al. (Document 9) and Martin et al. (Document 10)). However, its functional mechanism admits of several theories, and is yet to be established.
It has also been pointed out that SDF-1 plays an essential role in embryogenesis of the hematopoietic, cardiovascular, and nervous systems (e.g., Zou et al. (Document 11) and Tachibana et al. (Document 12)). On the other hand, many of the biological functions of SDF-1 in adult tissue are still unknown.
As described above, it is extremely important for advancement of the understanding of SDF-1 to develop a technique for accurately quantifying and monitoring SDF-1 in a biological fluid sample. It is needless to say that an accurate measurement method in biological fluid samples would similarly make academic and clinical contributions in other chemokines and cytokines as well. From this perspective, an assay method for detecting cytokines with a higher sensitivity is desired.